A highly specific phosphatase that acts on ADP-ribose 1″-phosphate, a metabolite of tRNA splicing in Saccharomyces cerevisiae

One molecule of ADP-ribose 1″,2″-cyclic phosphate (Appr>p) is formed during each of the approximately 500 000 tRNA splicing events per Saccharomyces cerevisiae generation. The metabolism of Appr>p remains poorly defined. A cyclic phosphodiesterase (Cpd1p) has been shown to convert Appr>p to ADP-ribose-1″-phosphate (Appr1p). We used a biochemical genomics approach to identify two yeast phosphatases that can convert Appr1p to ADP-ribose: the product of ORF YBR022w (now Poa1p), which is completely unrelated to other known phosphatases; and Hal2p, a known 3′-phosphatase of 5′,3′-pAp. Poa1p is highly specific for Appr1p, and thus likely acts on this molecule in vivo. Poa1 has a relatively low K(M) for Appr1p (2.8 μM) and a modest k(cat) (1.7 min(−1)), but no detectable activity on several other substrates. Furthermore, Poa1p is strongly inhibited by ADP-ribose (K(I), 17 μM), modestly inhibited by other nucleotides containing an ADP-ribose moiety and not inhibited at all by other tested molecules. In contrast, Hal2p is much more active on pAp than on Appr1p, and several other tested molecules were Hal2p substrates or inhibitors. poa1-Δ mutants have no obvious growth defect at different temperatures in rich media, and analysis of yeast extracts suggests that ∼90% of Appr1p processing activity originates from Poa1p

Secondary structure models of the 3′ untranslated regions of diverse R2 RNAs

The RNA structure of the 3′ untranslated region (UTR) of the R2 retrotransposable element is recognized by the R2-encoded reverse transcriptase in a reaction called target primed reverse transcription (TPRT). To provide insight into structure–function relationships important for TPRT, we have created alignments that reveal the secondary structure for 22 Drosophila and five silkmoth 3′ UTR R2 sequences. In addition, free energy minimization has been used to predict the secondary structure for the 3′ UTR R2 RNA of Forficula auricularia. The predicted structures for Bombyx mori and F. auricularia are consistent with chemical modification data obtained with β-ethoxy-α-ketobutyraldehyde (kethoxal), dimethyl sulfate, and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate. The structures appear to have common helices that are likely important for function

Platelet transfusion—the new immunology of an old therapy

Platelet transfusion has been a vital therapeutic approach in patients with hematologic malignancies for close to half a century. Randomized trials show that prophylactic platelet transfusions mitigate bleeding in patients with acute myeloid leukemia. However, even with prophylactic transfusions, as many as 75% of patients experience hemorrhage. While platelet transfusion efficacy is modest, questions and concerns have arisen about the risks of platelet transfusion therapy. The acknowledged serious risks of platelet transfusion include viral transmission, bacterial sepsis, and acute lung injury. Less serious adverse effects include allergic and non-hemolytic febrile reactions. Rare hemolytic reactions have occurred due to a common policy of transfusing without regard to ABO type. In the last decade or so, new concerns have arisen; platelet derived lipids are implicated in transfusion related acute lung injury after transfusion. With the recognition that platelets are immune cells came the discoveries that supernatant IL-6, IL-27 sCD40L, and OX40L are closely linked to febrile reactions and sCD40L with acute lung injury. Platelet transfusions are pro-inflammatory, and may be pro-thrombotic. Anti-A and anti-B can bind to incompatible recipient or donor platelets and soluble antigens, impair hemostasis and thus increase bleeding. Finally, stored platelet supernatants contain biological mediators such as VEGF and TGF-β1 that may compromise the host versus tumor response. This is particularly of concern in patients receiving many platelet transfusions, as for acute leukemia. New evidence suggests that removing stored supernatant will improve clinical outcomes.This new view of platelets as pro-inflammatory and immunomodulatory agents suggests that innovative approaches to improving platelet storage and pre-transfusion manipulations to reduce toxicity could substantially improve the efficacy and safety of this long employed therapy

Breaking the mold: transcription factors in the anucleate platelet and platelet-derived microparticles

Platelets are small anucleate blood cells derived from megakaryocytes. In addition to their pivotal roles in hemostasis, platelets are the smallest, yet most abundant, immune cell and regulate inflammation, immunity, and disease progression. Although platelets lack DNA, and thus no functional transcriptional activities, they are nonetheless rich sources of RNAs, possess an intact spliceosome, and are thus capable of synthesizing proteins. Previously, it was thought that platelet RNAs and translational machinery were remnants from the megakaryocyte. We now know that the initial description of platelets as cellular fragments is an antiquated notion, as mounting evidence suggests otherwise. Therefore, it is reasonable to hypothesize that platelet transcription factors are not vestigial remnants from megakaryoctes, but have important, if only partly understood functions. Proteins play multiple cellular roles to minimize energy expenditure for maximum cellular function; thus, the same can be expected for transcription factors. In fact, numerous transcription factors have non-genomic roles, both in platelets and in nucleated cells. Our lab and others have discovered the presence and nongenomic roles of transcription factors in platelets, such as the nuclear factor kappa β (NFκB) family of proteins and peroxisome proliferator activated receptor gamma (PPARγ). In addition to numerous roles in regulating platelet activation, functional transcription factors can be transferred to vascular and immune cells through platelet microparticles. This method of transcellular delivery of key immune molecules may be a vital mechanism by which platelet transcription factors regulate inflammation and immunity. At the very least, platelets are an ideal model cell to dissect out the nongenomic roles of transcription factors in nucleated cells. There is abundant evidence to suggest that transcription factors in platelets play key roles in regulating inflammatory and hemostatic functions